Loss control of Mcm5 interaction with chromatin in cdc6-1 mutated in CDC-NTP motif.
Loss control of Mcm5 interaction with chromatin in cdc6-1 mutated in CDC-NTP motif.
复制标题
Mcm5 与 CDC-NTP 基序突变的 cdc6-1 染色质相互作用失去控制。
DOI：
10.1089/10445490050085933
复制
发表时间：
2000
影响因子：
3.1
通讯作者：
Jong,A
中科院分区：
文献类型：
--
作者：
Feng,L;Hu,Y;Wang,B;Wu,L;Jong,A
SaccharomycescleiaeCdc 6通过与起始识别复合物（originrecognitioncomplex，ORC）相互作用，结合到细胞内，在复制前复合物（pre-replicativecomplex，pre-RC）的建立和维持中发挥重要作用 与染色质起源有关。这些相互作用是将微型染色体维持蛋白（MCMs）和其他起始蛋白加载到复制起点上所必需的。虽然温度敏感的ecdc 6突变体cdc 6 -1已被广泛用于这些研究，但cdc 6 - 1突变的分子机制尚不清楚。在这项研究中，我们已经确定了一个碱基取代Gly 260 → Asp，附近的CDC-NTP基序。用染色质免疫沉淀法（CHIP）检测发现cdc 6 - 1不能将Mcm 5负载到复制起点上。染色质组分用于研究 野生型和突变体背景中的mcm 5结合。这些研究表明，Cdc 6也参与从染色质卸载Mcm 5。具体而言，cdc 6 - 1突变蛋白cdc 6（G260 D）， 其不能将Mcm 5装载到复制起点上，也不能卸载Mcm 5蛋白。此外，野生型CDC 6的过表达加速了Mcm 5从染色质组分中的卸载。 在没有functionalCdc 6的情况下，Mcm 5蛋白表现出与染色质的非起源结合，细胞周期停滞在G1 S期。我们的研究结果表明cdc 6（G260 D） 突变蛋白不能组装可操作的复制复合物，野生型Cdc 6通过控制MCMs从染色质起点的卸载而在防止再复制中起作用。
Saccharomyces cerevisiaeCdc6 plays an essential role in establishing and maintaining the prereplicative complex (pre-RC) by interacting with the origin recognition complex (ORC) and associating with chromatin origins. These interactions are required to load minichromosome maintenance proteins (MCMs) and other initiator proteins onto replication origins. Although the temperature-sensitivecdc6mutant,cdc6-1, has been widely used for these studies, the molecular mechanism of thecdc6-1mutation has been unclear. In this study, we have identified a base substitution at Gly260→ Asp, near the CDC-NTP motif. Using a chromatin immunoprecipitation assay (CHIP), we found thatcdc6-1fails to load Mcm5 onto the replication origins. Chromatin fractions were used to study Mcm5 binding in both the wildtype and mutant background. These studies indicated that Cdc6 is also involved in unloading Mcm5 from chromatin. Specifically, thecdc6-1mutation protein, cdc6(G260D), which failed to load Mcm5 onto replication origins, also failed to unload the Mcm5 protein. Furthermore, the overexpression of wildtypeCDC6accelerated the unloading of Mcm5 from chromatin fractions. In the absence of functionalCdc6, the Mcm5 protein showed nonorigin binding to chromatin with the cell cycle arrested at the G1S phase transition. Our results suggested that the cdc6(G260D) mutant protein fails to assemble an operational replicative complex and that wildtype Cdc6 plays a role in preventing re-replication by controlling the unloading the MCMs from chromatin origins.